U.S. patent number 10,221,389 [Application Number 15/230,788] was granted by the patent office on 2019-03-05 for method of producing cell population with high target cell purity.
This patent grant is currently assigned to TERUMO KABUSHIKI KAISHA. The grantee listed for this patent is TERUMO KABUSHIKI KAISHA. Invention is credited to Eri Noguchi.
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United States Patent |
10,221,389 |
Noguchi |
March 5, 2019 |
Method of producing cell population with high target cell
purity
Abstract
A method is disclosed of producing a cell population with a high
target cell purity, including changing a character of contaminating
cells in a cell population including target cells and the
contaminating cells, and removing the contaminating cells and/or
collecting the target cells based on the changed character. The
disclosure also relates to a cell population with a high target
cell purity, produced by the method, and a method of producing a
sheet-shaped cell culture, including culturing the cell population
into a sheet shape, a sheet-shaped cell culture containing the cell
population, a medical composition containing an effective component
selected from the group including the cell population and the
sheet-shaped cell culture, and a method of treating a disease in a
subject, including administering an effective amount of the cell
population, the sheet-shaped cell culture or the medical
composition, to the subject needing the same.
Inventors: |
Noguchi; Eri (Kanagawa,
JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
TERUMO KABUSHIKI KAISHA |
Shibuya-ku, Tokyo |
N/A |
JP |
|
|
Assignee: |
TERUMO KABUSHIKI KAISHA
(Shibuya-Ku, Tokyo, JP)
|
Family
ID: |
54009079 |
Appl.
No.: |
15/230,788 |
Filed: |
August 8, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160340643 A1 |
Nov 24, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/JP2015/055467 |
Feb 25, 2015 |
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Foreign Application Priority Data
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Feb 26, 2014 [JP] |
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2014-035358 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
43/00 (20180101); A61L 27/3834 (20130101); A61K
35/28 (20130101); C12N 5/0658 (20130101); A61P
9/04 (20180101); A61P 9/10 (20180101); C12N
5/0081 (20130101); A61K 35/34 (20130101) |
Current International
Class: |
A61K
35/28 (20150101); A61L 27/38 (20060101); A61K
35/34 (20150101); C12N 5/00 (20060101); C12N
5/077 (20100101) |
References Cited
[Referenced By]
U.S. Patent Documents
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Feb 2003 |
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Feb 2003 |
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2004-508824 |
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Mar 2004 |
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JP |
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2007-89442 |
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Apr 2007 |
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JP |
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2007-528755 |
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Oct 2007 |
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JP |
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2010-081829 |
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Apr 2010 |
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JP |
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2010-81829 |
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2011-110368 |
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Jun 2011 |
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Jun 2011 |
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2011-115058 |
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Jun 2011 |
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JP |
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WO 2005/011524 |
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Feb 2005 |
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WO |
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WO 2006/080434 |
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WO |
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WO 2012/118099 |
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Sep 2012 |
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WO |
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WO 2014/185517 |
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Nov 2014 |
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WO |
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Primary Examiner: Fox; Allison M
Attorney, Agent or Firm: Buchanan Ingersoll & Rooney
PC
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of International Application No.
PCT/JP2015/055467 filed on Feb. 25, 2015, which claims priority to
JP 2014-035358 filed on Feb. 26, 2014, the entire contents of which
are incorporated herein by reference.
Claims
What is claimed is:
1. A method of producing a cell population with a high target cell
purity, comprising: changing a character of contaminating cells in
a cell population including target cells and the contaminating
cells, the target cells being skeletal myoblasts, the contaminating
cells being fibroblasts, and wherein the character is selected from
the group consisting of cell shape, size, specific gravity, surface
charge, adhesion potency, and marker, wherein the changing of the
character of the contaminating cells comprises: converting the
fibroblasts into fat cells, megakaryocytes, platelets,
hepatocyte-like cells, macrophage-like cells, neurons, or cardiac
muscle cells; and separating the fibroblasts from the skeletal
myoblasts based on a difference in the cell size or the specific
gravity of the fibroblasts and the skeletal myoblasts; removing the
contaminating cells and/or collecting the target cells based on the
changed characters; and culturing the target cells into a sheet
shape.
2. The method according to claim 1, further comprising: performing
a pretreatment, the pretreatment including a filtration, a
centrifugation, or a separation between adhered cells and floating
cells by an adhesion culture to separate the skeletal myoblasts and
the fibroblasts from a tissue collected from a living body before
the changing of the character of the contaminating cells in the
cell population.
3. A method of producing a cell population with a high target cell
purity, comprising: changing a character of contaminating cells in
a cell population including target cells and the contaminating
cells, the target cells being skeletal myoblasts, the contaminating
cells being fibroblasts, and wherein the character is selected from
the group consisting of cell shape, size, specific gravity, surface
charge, adhesion potency, and marker, wherein the changing of the
character of the contaminating cells comprises: converting the
fibroblasts into fat cells; and separating the fibroblasts from the
skeletal myoblasts based on a difference in the cell size or the
specific gravity of the fibroblasts and the skeletal myoblasts;
removing the contaminating cells and/or collecting the target cells
based on the changed characters; and culturing the target cells
into a sheet shape.
4. A method of producing a sheet-shaped cell culture, the method
comprising: changing a character of contaminating cells in a cell
population including target cells and the contaminating cells that
is collected from a subject, the target cells being skeletal
myoblasts, the contaminating cells being fibroblasts, and wherein
the character is selected from the group consisting of cell shape,
size, specific gravity, surface charge, adhesion potency, and
marker, wherein the changing of the character of the contaminating
cells comprises: converting the fibroblasts into fat cells,
megakaryocytes, platelets, hepatocyte-like cells, macrophage-like
cells, neurons, or cardiac muscle cells; and separating the
fibroblasts from the skeletal myoblasts based on a difference in
the cell size or the specific gravity of the fibroblasts and the
skeletal myoblasts; obtaining a cell population with a high target
cell purity by removing the contaminating cells and/or collecting
the target cells based on the changed characters; freezing the cell
population; thawing the frozen cell population; culturing the cell
population into a sheet shape to forma sheet-shaped cell culture;
and recovering the sheet-shaped cell culture formed.
5. The method according to claim 4, further comprising: performing
a pretreatment, the pretreatment including a filtration, a
centrifugation, or a separation between adhered cells and floating
cells by an adhesion culture to separate the skeletal myoblasts and
the fibroblasts from a tissue collected from a living body before
the changing of the character of the contaminating cells in the
cell population.
6. A method of producing a sheet-shaped cell culture, the method
comprising: changing a character of contaminating cells in a cell
population including target cells and the contaminating cells that
is collected from a subject, the target cells being skeletal
myoblasts, the contaminating cells being fibroblasts, and wherein
the character is selected from the group consisting of cell shape,
size, specific gravity, surface charge, adhesion potency, and
marker, wherein the changing of the character of the contaminating
cells comprises: converting the fibroblasts into fat cells,
megakaryocytes, platelets, hepatocyte-like cells, macrophage-like
cells, neurons, or cardiac muscle cells; and separating the
fibroblasts from the skeletal myoblasts based on a difference in
the cell size or the specific gravity of the fibroblasts and the
skeletal myoblasts; obtaining a cell population with a high target
cell purity by removing the contaminating cells and/or collecting
the target cells based on the changed characters; freezing the cell
population; thawing the frozen cell population; culturing the cell
population into a sheet shape to forma sheet-shaped cell culture;
and recovering the sheet-shaped cell culture formed.
7. A method of producing a cell population with a high target cell
purity, comprising: changing a character of contaminating cells in
a cell population including target cells and the contaminating
cells, the target cells being skeletal myoblasts, the contaminating
cells being fibroblasts, and wherein the character is selected from
the group consisting of cell size or specific gravity; removing the
contaminating cells and/or collecting the target cells based on the
changed character; and culturing the target cells into a sheet
shape.
8. The method according to claim 7, wherein the changing of the
character of the contaminating cells comprises: converting the
fibroblasts into megakaryocytes, platelets, hepatocyte-like cells,
macrophage-like cells, neurons, or cardiac muscle cells; and
separating the fibroblasts from the skeletal myoblasts based on a
difference in the cell size of the fibroblasts and the skeletal
myoblasts.
9. The method according to claim 7, wherein the changing of the
character of the contaminating cells comprises: converting the
fibroblasts into fat cells; and separating the fibroblasts from the
skeletal myoblasts based on a difference in the specific gravity of
the fibroblasts and the skeletal myoblasts.
Description
TECHNICAL FIELD
The present disclosure relates to a method of producing a cell
population with a high target cell purity, a cell population
obtained by the method, and a medical composition containing the
cell population.
BACKGROUND
In recent years, attempts to transplant various cells have been
conducted in order to repair injured tissue. For instance, for
repairing cardiac muscle tissue injured due to ischemic cardiopathy
such as stenocardia and myocardial infarction, attempts have been
made to utilize fetal cardiac muscle cells, skeletal myoblasts,
mesenchymal stem cells, cardiac stem cells, and ES cells. (See
Haraguchi et al., Stem Cells Transl Med. 2012 February; 1(2):
136-41).
As part of such attempts, cell structures formed by utilizing a
scaffold and sheet-shaped cell cultures obtained by forming cells
in a sheet shape have been developed (See JP-T-2007-528755).
In regard of application of a sheet-shaped cell culture to therapy,
investigations of utilization of a cultured skin sheet for a skin
injury due to burn or the like, utilization of a sheet-shaped cell
culture of corneal epithelium for a corneal injury, utilization of
a sheet-shaped cell culture of oral mucosa for endoscopic resection
of esophageal cancer have been under way.
Cells to be used for cell transplantation are normally obtained by
separating them from the subject's tissue relevant to the
transplantation, in order to avoid adverse events such as
rejection. In many cases, however, the tissue includes other cells
than the target cells. If the cells obtained from the tissue are
cultured without purification of the target cells, the other cells
than the target cells are also proliferated, lowering the
proportion of the target cells in the cells to be used for
transplantation. However, when the other cells than the target
cells are transplanted, a therapeutic effect as high as that in the
case of transplantation of the target cells cannot be obtained. It
may be impossible, therefore, to obtain a sufficient therapeutic
effect even by transplanting the cells obtained from the tissue, if
the cells to be transplanted are low in target cell purity.
As a measure to enhance the proportion of the target cells
contained in a cell population separated from the tissue, there has
been known, for example, a method wherein in separating skeletal
myoblasts from a skeletal muscle tissue, the skeletal muscle tissue
is subjected to an enzyme treatment by immersion in a proteinase
solution for a predetermined time, the resulting enzyme treatment
liquid is discarded, thereafter the skeletal muscle tissue is again
subjected to an enzyme treatment by immersion in an proteinase
solution for a predetermined time, and cells contained in the
enzyme treatment liquid thus obtained are recovered
(JP-A-2007-89442).
SUMMARY
A method is disclosed of producing a cell population with a high
target cell purity from a cell population including target cells
and contaminating cells, and more particularly a method of
producing such a cell population easily and efficiently, a cell
population with a high target cell purity obtained by the method,
and a medical composition containing the cell population.
In accordance with an exemplary embodiment, when a character of
contaminating cells in a cell population including target cells and
the contaminating cells is changed, and the contaminating cells are
removed and/or the target cells are collected on the basis of the
changed character, a cell population can be obtained with a high
target cell purity.
A method is disclosed of producing a cell population with a high
target cell purity, including: changing a character of
contaminating cells in a cell population including target cells and
the contaminating cells; and removing the contaminating cells
and/or collecting the target cells, on the basis of the changed
character.
According to an exemplary embodiment, the contaminating cells are
fibroblasts.
According to an exemplary embodiment, the character is selected
from the group including cell shape, size, specific gravity,
surface charge, adhesion potency, and marker.
According to an exemplary embodiment, the target cells are skeletal
myoblasts or mesenchymal stem cells.
According to an exemplary embodiment, a cell population with a high
target cell purity, produced by the method as disclosed herein.
According to an exemplary embodiment, a method of producing a
sheet-shaped cell culture, which includes culturing the cell
population into a sheet shape.
According to an exemplary embodiment, wherein the sheet-shaped cell
culture, including the cell population as disclosed herein.
According to an exemplary embodiment, a medical composition
containing an effective component selected from the group including
the cell population and the sheet-shaped cell culture as disclosed
herein.
According to an exemplary embodiment, the medical composition as
disclosed herein for treating a disease associated with tissue
abnormality.
According to an exemplary embodiment, a method of treating a
disease in a subject, including administering an effective amount
of the cell population, the sheet-shaped cell culture or the
medical composition as disclosed herein, to the subject needing the
same.
According to the present disclosure, the purity of target cells
included in a cell population that is collected from a living body
can be enhanced and includes a plurality of kinds of cells, to
enhance the therapeutic effect of cell transplantation, and to make
efficient the production of a cell composition for use in such a
therapy. In addition, by selecting cell size, specific gravity, or
surface charge as the character to be changed, a technique is
disclosed which is simple and capable of treating a large amount of
cells at a time, such as filtration and centrifugation, and whereby
target cells can be efficiently purified.
A method is disclosed of producing a sheet-shaped cell culture, the
method comprising: changing a character of contaminating cells in a
cell population including target cells and the contaminating cells
that is collected from a subject; obtaining a cell population with
a high target cell purity by removing the contaminating cells
and/or collecting the target cells based on the changed character;
freezing the cell population; thawing the frozen cell population;
culturing the cell population into a sheet shape to form a
sheet-shaped cell culture; and recovering the sheet-shaped cell
culture formed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing a fundamental concept of the present
disclosure.
DETAILED DESCRIPTION
Unless defined otherwise herein, all the technical terms and
scientific terms used herein have the same meanings as normally
understood by persons skilled in the art. All the patents, patent
applications, published patent applications and other publications
referenced herein are quoted herein in their entireties by
reference.
A method is disclosed of producing a cell population with a high
target cell purity, including (1) a step of changing a character of
contaminating cells in a cell population including target cells and
the contaminating cells, and (2) a step of removing the
contaminating cells and/or collecting the target cells based on the
changed character (hereinafter the method may be referred to simply
as "cell population production method").
In the present disclosure, the target cells and the contaminating
cells can be arbitrarily set according, for example, to the use of
the cell population obtained. Since the cell population including
the target cells and the contaminating cells is typically prepared
from a tissue collected from a living body, the target cells and
the contaminating cells are typically selected from the cells
present in the same tissue. For example, a cell population prepared
from a skeletal muscle tissue is composed mainly of skeletal
myoblasts and fibroblasts, one of them may be set as the target
cells and the other may be set as the contaminating cells. Thus, in
one embodiment of the present disclosure, the target cells are
skeletal myoblasts and the contaminating cells are fibroblasts. In
another embodiment of the present disclosure, the target cells are
fibroblasts and the contaminating cells are skeletal myoblasts. A
cell population prepared from bone marrow tissue is composed mainly
of mesenchymal stem cells, hematopoietic stem cells, blood
precursor cells, and fibroblasts, one or more of them may be set as
the target cells, and the other or others may be set as the
contaminating cells. Thus, in one embodiment of the present
disclosure, examples of a setting that may be adopted include,
without restriction, a setting wherein the target cells are
mesenchymal stem cells and the contaminating cells are
hematopoietic stem cells, blood precursor cells and fibroblasts,
and a setting wherein the target cells are mesenchymal stem cells
and hematopoietic stem cells and the contaminating cells are blood
precursor cells and fibroblasts.
The target cells and the contaminating cells can also be selected
from part of cell species present in body tissues. In other words,
it is unnecessary for all the cell species present in body tissues
to be classified into the target cells and the contaminating cells
in the present disclosure. A method can be adopted wherein cell
species that can be easily separated from the target cells without
changing a character thereof are preliminarily removed by a
pretreatment, and the target cells and the contaminating cells are
selected from the cell species remaining after the pretreatment.
Examples of the pretreatment applicable here include, without
restriction, filtration (for example, filtration by filter or
microchannel device), centrifugation (for example, density gradient
centrifugation, isodensity centrifugation, countercurrent
centrifugal elutriation (CCE), etc.), and separation between
adhered cells and floating cells by adhesion culture. In accordance
with an exemplary embodiment, for example, in the case of producing
a cell population with a high purity of mesenchymal stem cells as
target cells from bone marrow tissue, floating cells (for example,
hematopoietic stem cells, blood precursor cells, hematocyte-based
cells, etc.) can be removed by a pretreatment such as adhesion
culture, and fibroblasts that are contained in the remaining
adhered-cell population and are difficult to separate from the
mesenchymal stem cells can be selected as the contaminating cells
(namely, the cells whose character is to be changed) in the present
disclosure. Therefore, the cell population production method of the
present disclosure may further include a step of removing other
cells than the target cells and the contaminating cells from a
tissue collected from a living body before the step of changing the
character of the contaminating cells.
In the case of using a cell population for medical use (examples
thereof include, without restriction, such uses as treatment of
diseases, transplantation therapy, and regenerative therapy),
examples of cells from which the target cells can be selected
include, without restriction, hematopoietic stem cells for use in
treatment of hemopathy or the like, lymphocytes for use in
immunotherapy or the like, immunocytes such as dendritic cells,
cells capable of forming sheet-shaped cell cultures, for example,
myoblasts (for example, skeletal myoblasts), mesenchymal stem cells
(for example, those derived from bone marrow, adipose tissues,
peripheral blood, skin, hair roots, muscular tissues, endometria,
placentas, and cord blood), cardiac muscle cells, fibroblasts,
cardiac stem cells, embryonic stem cells, synovial cells,
chondrocytes, epithelial cells (for example, oral mucosal
epithelial cells, retinal pigment epithelial cells, nasal mucosa
epithelial cells, etc.), endothelial cells (for example, vascular
endothelial cells), hepatocytes (for example, hepatic parenchymal
cells), pancreatic cells (for example, pancreatic islet cells),
renal cells, adrenal cells, periodontal ligament cells, gingival
cells, periosteal cells, and skin cells. In this case, the
contaminating cells can be selected from other cells than the
target cells that are present in the tissue in which the target
cells exist, particularly, those cells that are difficult to
separate from the target cells and that are high in proliferation
potency and/or abundance ratio in the tissue.
In one embodiment of the present disclosure, the contaminating
cells are fibroblasts. In one embodiment of the present disclosure,
the target cells are myoblasts, preferably skeletal myoblasts. In a
preferred embodiment of the present disclosure, the target cells
are skeletal myoblasts and the contaminating cells are fibroblasts.
In another embodiment of the present disclosure, the target cells
are mesenchymal stem cells. In a preferred embodiment of the
present disclosure, the target cells are mesenchymal stem cells and
the contaminating cells are fibroblasts.
As the character of the contaminating cells that is to be changed,
one or more of any known characters can be selected. It can be
preferable, however, that the character is a character, which,
after changed, is useful for separation of the contaminating cells
from the target cells. Examples of such a character include,
without restriction, cell size, specific gravity, surface charge,
presence/absence of marker expression, adhesion potency,
proliferation potency, and chemotaxis. Changing of the character
can be carried out by any known technique or a combination of such
techniques. For instance, it is known that a cell species can be
converted into another cell species by introduction of a gene such
as a transcription factor or by administration of a drug (for
example, Nizzardo et al., Cell Transplant. 2013; 22(6): 921-44, Zhu
et al., Cell Reprogram. 2012 April; 14(2): 99-105, etc.).
Therefore, the step of changing a character of contaminating cells
in the present disclosure includes conversion of the contaminating
cells into cell species easy to separate from the target cells, the
conversion being carried out by such a technique as
just-mentioned.
For instance, techniques for conversion of fibroblasts into fat
cells, megakaryocytes, platelets, hepatocyte-like cells,
macrophage-like cells, neurons, cardiac muscle cells or the like
have been known (for example, the above-mentioned Nizzardo et al.,
2013, Zhu et al., 2012, Ono et al., Blood. 2012 Nov. 1; 120(18):
3812-21, etc.). Therefore, by conversion of fibroblasts as the
contaminating cells into any one of these cells which are different
in character from the desired target cells, for example, skeletal
myoblasts or mesenchymal stem cells, the converted contaminating
cells can be separated or removed from the skeletal myoblasts or
mesenchymal stem cells and/or the skeletal myoblasts or mesenchymal
stem cells can be separated or removed from the converted
contaminating cells. For instance, while the cell diameter of
skeletal myoblasts is approximately, for example, 11 to 18 .mu.m,
the cell diameter of megakaryocytes is approximately, for example,
35 to 160 .mu.m, the cell diameter of platelets is approximately,
for example, 2 to 4 .mu.m, the cell diameter of hepatocytes is
approximately, for example, 20 to 30 .mu.m, and the cell diameter
of macrophages is approximately, for example, 25 to 50 .mu.m. When
fibroblasts are converted into any of these cells, therefore, the
converted cells can be separated from the skeletal myoblasts by
utilizing the difference in cell size. In addition, fat cells are
different from skeletal myoblasts or mesenchymal stem cells in
specific gravity. When fibroblasts are converted into fat cells,
therefore, the converted cells can be separated from the skeletal
myoblasts or mesenchymal stem cells by utilizing the difference in
cell specific gravity.
Conversion of fibroblasts into fat cells can be conducted by a
method examples of which include, without restriction, introduction
of such a factor as PPAR.gamma. (Tontonoz et al., Cell. 1994 Dec.
30; 79(7): 1147-56), C/EBP.alpha. (Freytag et al., Genes Dev. 1994
Jul. 15; 8(14): 1654-63) or ADD1/SREBP-1 (Kim and Spiegelman, Genes
Dev. 1996 May 1; 10(9): 1096-107), and a treatment by a combination
of a TGF-.beta. signaling inhibitor (for example, TGF-.beta. RI
kinase inhibitor such as SB43152) with a ROCK signaling inhibitor
(for example, Thiazovivin) (the above-mentioned Zhu et al., 2012).
Conversion of fibroblasts into hepatocyte-like cells can be
performed by a method, examples of which can include, without
restriction, a combination of introduction of Gata4, Hnf1.alpha.
and Foxa3 with inactivation of p19Arf (Huang et al., Nature. 2011
May 11; 475 (7356): 386-9), and introduction of Hnf4.alpha.
together with Foxa1, Foxa2 or Foxa3 (Sekiya and Suzuki, Nature.
2011 Jun. 29; 475(7356): 390-3). Conversion of fibroblasts into
macrophage-like cells can be carried out by a method, examples of
which can include, without restriction, introduction of PU. 1
together with C/EBP.alpha. or C/EBP.beta. (Feng et al., Proc Natl
Acad Sci USA. 2008 Apr. 22; 105(16): 6057-62). Conversion of
fibroblasts into megakaryocytes and platelets can be conducted by a
method, examples of which can include, without restriction,
introduction of p45NF-E2, Maf G and Maf K (the above-mentioned Ono
et al., 2012). Conversion of fibroblasts into neurons can be
performed by a method, examples of which can include, without
restriction, introduction of Ascl1, Brn2 and Myt1I, introduction of
Ascl1, Brn2, Myt1I and NeuroD1, introduction of miR-9/9, miR-124,
NeuroD2, Ascl1 and Myt1I, introduction of Mash1, Nurr1 and Lmx1a,
introduction of Ascl1, Pitx3, Lmx1a, Nurr1, Foxa2 and EN1, and
introduction of Ascl1, Brn2, Nyt11, Lhx3, Hb9, Is11, Ngn2 and
NEUROD1 (above-mentioned Nizzardo et al., 2013). Conversion of
fibroblasts into cardiac muscle cells can be carried out by a
method, examples of which can include introduction of Gata4, Tbx5
and Mef2c, and introduction of Oct4, Sox2, Klf4 and c-Myc (the
above-mentioned Nizzardo et al., 2013).
Examples of other technique for changing a character of
contaminating cells can include, without restriction, expression of
a desired marker or inhibition of marker expression, and conversion
into iPS cells and subsequent inducing of differentiation into
desired cells. Examples of the desired marker include, without
restriction, cell surface markers, and fluorescent proteins such as
GFP. The marker expression and the inhibition of marker expression
can be performed by any known technique. Examples of a marker
expression technique can include introduction of a marker-coding
nucleic acid into the contaminating cells. The introduction of a
nucleic acid can be conducted by use of a method, examples of which
can include, without restriction a calcium phosphate method, a
lipofection method, an ultrasonic introduction method, an
electroporation method, a particle gun method, methods based on
utilization of a vector (for example, adenovirus vector,
adeno-associated virus vector or retrovirus vector), and a
microinjection method. Examples of a technique for inhibiting
marker expression include, without restriction, an RNAi method and
an antisense method. The RNAi method may include introduction of an
RNAi molecule into the contaminating cells, and the antisense
method may include introduction of an antisense nucleic acid into
the contaminating cells.
Conversion into iPS cells can be performed, for example, by
introduction of such a gene as OCT3/4, SOX2, KLF4, C-MYC, LIN28,
NANOG, and GLIS1 into cells (for example, a combination of OCT3/4,
SOX2, KLF4 and C-MYC, a combination of OCT3/4, SOX2, LIN28 and
NANOG, a combination of OCT3/4, SOX2, KLF4 and GLIS1, a combination
of OCT3/4, SOX2 and KLF4, a combination of OCT3/4 and SOX2, etc.).
Methods for conversion into iPS cells have been known in the art
(see, for example, Miyazaki et al., Jpn J Clin Oncol. 2012
September; 42(9): 773-9, Bayart and Cohen-Haguenauer, Curr Gene
Ther. 2013 April; 13(2): 73-92), and any known conversion method or
a modified method thereof can be used in the present
disclosure.
Methods for conversion from iPS cells into other cell species have
also been known in the art, and any known conversion method or a
modified method thereof can be used in the present disclosure.
Examples of a method known for conversion from iPS cells into
megakaryocytes or platelets include a method wherein cells are
cultivated sequentially in a culture medium containing STMspan-ACF,
BMP4, VEGF and bFGF, next in a culture medium containing STEM-diff
APEL Medium, TPO, SCF, Flt-3 ligand, IL-3, IL-6 and heparin, and
then in a culture medium containing STEMspan-ACF, TPO, SCF, IL-6,
IL-9 and heparin (Feng et al., Stem Cell Reports. 2014 Nov. 11;
3(5): 817-31). Examples of a method known for conversion from iPS
cells into hepatocyte-like cells include a method wherein cells are
cultivated sequentially in the presence of such factors as activin
A, Wnt3, FGF4, bFGF, BMP-2, KGF, HGF, and oncostatin M (Subba Rao
et al., World J Gastroenterol. 2013 Jun. 14; 19(22): 3385-96).
When used herein, an RNAi molecule refers to any molecule that
provides RNA interference, and include, without restriction,
double-stranded RNAs such as sRNA (small interfering RNA), miRNA
(micro RNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA),
piRNA (Piwi-interacting RNA), and rasiRNA (repeat associated sRNA)
and modified products thereof. These RNAi molecules are
commercially available or can be designed and produced based on
known sequence information. In addition, when used herein, an
antisense nucleic acid includes RNA, DNA, PNA and complexes
thereof.
When a nucleic acid (for example, a marker-coding nucleic acid, an
RNAi molecule or a nucleic acid for coding the same) or a drug is
applied to the contaminating cells, a delivery technique specific
to the contaminating cells can be used. Examples of such a delivery
technique include active targeting in which a targeting agent (for
example, targeting ligand) specific to the contaminating cells is
used (Marcucci and Lefoulon, Drug Discov Today. 2004 Mar. 1; 9(5):
219-28, Torchilin, Eur J Pharm Sci. 2000 October; 11 Suppl 2:
S81-91). There are known a large number of targeting agents that
are each specific to a specified cell. By the active targeting, a
nucleic acid or a drug can be made to act only on the contaminating
cells without acting on the target cells. Therefore, it is
possible, for example, to utilize a nucleic acid or drug that
produces the same effect (for example, conversion into the same
cell species) on both the target cells and the contaminating cells.
Examples of a targeting agent specific to fibroblasts include,
without restriction, substances that bind specifically to TE-7
(refer to, for example, Rosendaal et al., J Cell Sci. 1994 January;
107(Pt 1): 29-37, Goodpaster et al., J Histochem Cytochem. 2008
April; 56(4): 347-58, and the like) which is a cell surface marker
specific to fibroblasts, for example, an antibody for TE-7 or the
antibody's fragments including an antigen recognition site (for
example, F(ab')2, Fab', Fab, Fv, scFv fragments).
When a nucleic acid is used through expression in the contaminating
cells, the nucleic acid can be placed under the control of a
regulatory sequence specific to the contaminating cells, for
helping ensure an expression specific to the contaminating cells.
By use of such a technique, it is possible to allow expression of
the nucleic acid only in the contaminating cells while inhibiting
expression of the nucleic acid in the target cells. Therefore, for
example, a nucleic acid can be used that produces the same effect
(for example, conversion into the same cell species) on both the
target cells and the contaminating cells. There are known
regulatory sequences specific to a particular cell. Examples of the
regulatory sequence, which can be used for nucleic acid expression
specific to fibroblasts can include, without restriction, ST2 gene
proximal promoter (Iwahana et al., Eur J Biochem. 1999 September;
264(2): 397-406), .alpha.11 integrin promoter (Lu et al., Matrix
Biol. 2010 April; 29(3): 166-76), FSP1 gene promoter (Okada et al.,
Am J Physiol. 1998 August; 275(2 Pt 2): F306-14), and
pro.alpha.2(I) collagen enhancer (Bou-Gharios et al., J Cell Biol.
1996 September; 134(5): 1333-44). By a joint use of such a
regulatory sequence with the aforementioned active targeting, the
cell specificity can be further enhanced.
The character of contaminating cells to be changed may be one
character or two or more characters in combination. For instance,
in the case where the contaminating cells are converted into
another cell species, a plurality of characters may be changed
simultaneously. In addition, it is possible to effect expression of
a plurality of markers or to inhibit expressions of a plurality of
markers, simultaneously.
Removal of the contaminating cells or collection of the target
cells can be carried out by any known technique according to the
character in question. For example, in the case where the character
to be changed is cell size, the removal of the contaminating cells
or collection of the target cells can be performed by utilizing
filtration through an appropriately sized filter (for example,
nylon mesh filter) or a microchannel device or centrifugation.
Where the character to be changed is cell specific gravity, the
removal of the contaminating cells or collection of the target
cells can be carried out by utilizing centrifugation (for example,
density gradient centrifugation, isodensity centrifugation,
countercurrent centrifugal elutriation, etc.). Where the character
to be changed is a cell surface marker, the removal of the
contaminating cells or the collection of the target cells can be
performed by utilizing a flow cytometry method, or an affinity
separation method (for example, affinity column method, magnetic
cell separation method, immunopanning, etc.). Where the character
after the change is expression of a fluorescent protein, the
removal of the contaminating cells or the collection of the target
cells can be carried out, for example, by utilizing a flow
cytometry method. Where the character to be changed is surface
charge, the removal of the contaminating cells or the collection of
the target cells can be performed, for example, by utilizing ion
exchange chromatography, electrophoresis, or dielectrophoresis.
Among these, preferred is a change of a character selected from the
group including cell size, specific gravity, and surface charge,
from the viewpoints of simplicity of the purifying operation and
the largeness of the amount of cells that can be purified at a
time.
The above-mentioned technique may be applied once or may be applied
multiple times, for the same sample. In addition, where two or more
techniques are present for the same character, only one of the
characters may be applied, or two or more characters may be applied
in combination. Furthermore, where a plurality of characters to be
changed are present, removal of the contaminating cells or
collection of the target cells may be performed while paying
attention to one of the characters or while paying attention to two
or more of the characters.
The expression "with a high target cell purity" in the present
disclosure means a target cell purity higher than the target cell
purity in natural tissues. For example, in the case where the
target cell purity in natural tissues is, for example, 50%, the
target cell purity after the method of the disclosure is carried
out may be, for example, not less than 60%, not less than 65%, not
less than 70%, not less than 75%, not less than 80%, not less than
85%, not less than 90%, not less than 95%, not less than 98% or not
less than 99%, and most preferably 100%. The target cell purity can
be determined by any known method. Examples of such a method can
include a method in which the target cells are labeled with an
antibody specific to the target cells, and the number of positive
cells to which the antibody is bound is divided by a total number
of the cells counted. The counting of the cells can be carried out
by microscopic observation of a sample dyed with a specific
antibody, image analysis of a microscopic image, or flow cytometric
analysis of a cell population dyed with a specific antibody. In the
case where the target cells are skeletal myoblasts, examples of a
marker specific to the cell include, without restriction, CD56,
.alpha.7 integrin, myosin heavy chain IIa, myosin heavy chain IIb,
myosin heavy chain IId (IIx), MyoD, MyfS, and myogenin. Where the
target cells are mesenchymal stem cells, examples of a marker
specific to the cell can include, without restriction, CD29, CD73,
CD90, CD105, and CD166.
The cell population production method of the present disclosure may
further include a step of culturing the collected target cells and
a step of subculturing the cultured cells. The cell cultivation and
subculture can be carried out by use of any known method. In
addition, the cell population production method of the present
disclosure may further include a step of introducing a gene into
the collected cells. The gene to be introduced is not particularly
limited so long as the gene is useful for therapy of a disease to
be treated, and may be, for example, a cytokine such as HGF. The
introduction of a gene can be performed by use of any known method
such as a calcium phosphate method, a lipofection method, an
ultrasonic introduction method, an electroporation method, a
particle gun method, methods utilizing a vector such as adenovirus
vector, retrovirus vector, etc., and a microinjection method.
Another aspect of the present disclosure relates to a cell
population with a high target cell purity produced by the cell
population production method of the present disclosure (hereinafter
this cell population may be referred to simply as "high-purity cell
population"). The high-purity cell population of the present
disclosure is obtained by the cell population production method of
the present disclosure, and has a high target cell purity. The
extent to which the target cell purity is high is as described
above. The high-purity cell population of the present disclosure
can include the target cells in a high proportion and is therefore
useful as a source of cells for use in medical use or the like. The
high-purity cell population of the present disclosure is preferably
aseptic. In addition, the high-purity cell population of the
present disclosure may be in adhesion to a culture vessel, may be
floating in a liquid that is physiologically acceptable, or may be
in a cryopreserved state.
A further aspect of the present disclosure relates to a method of
producing a sheet-shaped cell culture that can include a step of
culturing the high-purity cell population of the present disclosure
in a sheet shape (hereinafter, the method may be referred to simply
as "sheet-shaped cell culture production method").
In the present disclosure, the "sheet-shaped cell culture" refers
to a cell culture in which cells are interconnected to form a
sheet-shaped body, and is typically one composed of a single cell
layer, but includes those composed of two or more cell layers. The
cells may be interconnected directly (inclusive of the case where
the cells are interconnected through a cell element such as an
adhesion molecule) and/or through an intermediate substance. The
intermediate substance is not specifically restricted so long as it
is a substance capable of interconnecting the cells at least
physically (mechanically). The intermediate substance may be, for
example, an extracellular matrix. The intermediate substance is
preferably one derived from cells, particularly one derived from
the cells constituting the sheet-shaped cell culture. While the
cells are interconnected at least physically (mechanically), they
may further be interconnected functionally, for example, chemically
or electrically.
The sheet-shaped cell culture of the present disclosure preferably
does not include a scaffold (support), from the viewpoint of high
biocompatibility and a high therapeutic effect. A scaffold is
sometimes used in this technical field for adhering cells onto a
surface thereof and/or to the inside thereof in order to maintain
physical integrity of the sheet-shaped cell culture, and known
examples of the scaffold include films made of polyvinylidene
difluoride (PVDF). However, the sheet-shaped cell culture of the
present disclosure preferably can maintain its physical integrity
even without such a scaffold. The sheet-shaped cell culture of the
present disclosure, preferably, is composed only of a substance or
substances derived from the cells constituting the sheet-shaped
cell culture, and does not include other substances.
The step of culturing the high-purity cell population of the
present disclosure in a sheet shape can be performed by any known
technique. Examples of such a technique include, without
restriction, those described in Patent Document 1,
JP-A-2010-081829, JP-A-2010-226962, JP-A-2010-226991,
JP-A-2011-110368, JP-A-2011-115058, and JP-A-2011-172925. The step
of culturing the high-purity cell population in a sheet shape may
include a step of seeding the cell population to a culture
substrate and a step of culturing the seeded cell population into a
sheet shape.
The culture substrate is not particularly limited so long as the
cell population can form a sheet-shaped cell culture on the culture
substrate. Examples of the culture substrate include vessels formed
from various materials, and solid or semisolid surfaces in the
vessels. The vessel preferably has such a structure and material
that liquid such as a culture liquid is not permeated therethrough.
Examples of such a material include, without restriction,
polyethylene, polypropylene, Teflon.RTM., polyethylene
terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl
alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide,
polydimethylacrylamide, and metals (for example, iron, stainless
steel, aluminum, copper, brass). In addition, the vessel preferably
has at least one flat surface. Examples of such a vessel include,
without restriction, cell culture dishes, and cell culture bottles.
In addition, the vessel may have a solid or semisolid surface in
the inside thereof. Examples of the solid surface include plates
and vessels of such materials as above-mentioned, and examples of
the semisolid surface include gels and soft polymer matrixes. The
culture substrate may be produced by use of the above-mentioned
material, or a commercially available culture substrate may be
utilized. Examples of a preferable culture substrate include,
without restriction, those substrates having adhesive surfaces
suited to formation of the sheet-shaped cell culture thereon.
Specific examples include substrates having hydrophilic surfaces
such as the substrates whose surfaces are coated with corona
discharge-treated polystyrene, collagen gel or a hydrophilic
compound such as a hydrophilic polymer, and, further, substrates
whose surfaces are coated with an extracellular matrix such as
collagen, fibronectin, laminin, vitronectin, proteoglycan,
glycosaminoglycan, etc. or a cell adhesion factor such as cadherin
family, selectin family, integrin family, etc. Such substrates are
commercially available (for example, Corning.RTM. TC-Treated
Culture Dish, Corning).
The culture substrate may have a surface thereof coated with a
material whose property is varied in response to a stimulus, for
example, temperature or light. Examples of materials which can be
used as such a material include, without restriction, known
temperature-responsive materials composed of homopolymer or
copolymer of (meth)acrylamide compounds, N-alkyl-substituted
(meth)acrylamide derivatives (for example, N-ethylacrylamide,
N-n-propylacrylamide, N-n-propylmethacrylamide,
N-isopropylacrylamide, N-isopropylmethacrylamide,
N-cyclopropylacrylamide, N-cyclopropylmethacrylamide,
N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide,
N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide,
etc.), N,N-dialkyl-substituted (meth)acrylamide derivatives (for
example, N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide,
N,N-diethylacrylamide, etc.), (meth)acrylamide derivatives having a
cyclic group (for example, 1-(1-oxo-2-propenyl)-pyrrolidine,
1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine,
1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine,
1-(1-oxo-2-methyl-2-propenyl)-piperidine,
4-(1-oxo-2-methyl-2-propenyl)-morpholine, etc.), or vinyl ether
derivatives (for example, methyl vinyl ether), and known
photoresponsive materials such as light-absorbing polymers having
an azobenzene group, copolymers of a vinyl derivative of
triphenylmethane leucohydroxide with an acrylamide monomer, and
N-isopropylacrylamide gel containing spirobenzopyran (refer to, for
example, JP-A-2-211865, JP-A-2003-33177). By giving a predetermined
stimulus to these materials, it is possible to change, for example,
the hydrophilicity or hydrophobicity of the material and thereby to
promote exfoliation of the sheet-shaped cell culture adhered onto
the material. Culture dishes coated with a temperature-responsive
material are commercially available (for example, UpCell.RTM. from
CellSeed Inc.), and these dishes can be used in the production
method of the present disclosure.
The seeding of the cell population to the culture substrate can be
performed by any known technique and under any known conditions.
The seeding of the cell population to the culture substrate may be
conducted, for example, by injecting into a culture substrate
(culture vessel) a cell suspension obtained by suspending the cell
population in a culture liquid. For injection of the cell
suspension, there can be used an instrument suited to a cell
suspension injecting operation, such as a dropping pipette or a
pipette.
The step of culturing the seeded cell population into a sheet shape
can also be carried out by any known technique and under any known
conditions. Nonlimitative examples of such a technique are
described in Patent Document 1, JP-A-2010-081829, JP-A-2010-226962,
JP-A-2010-226991, JP-A-2011-110368, JP-A-2011-115058, and
JP-A-172925. Culturing the cell population into a sheet shape is
considered to be achieved by adhesion of cells to one another
through an intercellular adhesion mechanism such as adhesion
molecules and an extracellular matrix. Therefore, the step of
culturing the seeded cells into a sheet shape can be achieved, for
example, by culturing the cell population under a condition where
intercellular adhesion is formed. Such a condition may be any one
that enables formation of intercellular adhesion; normally,
intercellular adhesion can be formed in the same conditions as
general cell cultivation conditions. A person skilled in the art
can select an optimum condition according to the kind of the cell
population seeded. Herein the cultivation for culturing the seeded
cell population into a sheet shape may be referred to as
"sheet-formation cultivation."
The cell culture liquid for use in cultivation (which may be
referred to simply as "culture liquid" or "culture medium") is not
specifically restricted so long as it can maintain the existence of
cells, and, those containing amino acids, vitamins and electrolytes
as main components can be utilized. As the culture liquid, there
can be used a culture liquid based on a basal culture medium for
cell cultivation. Examples of such a basal culture medium include,
without restriction, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102,
104, 107, 120, 131, 153, 199, etc.), F12/DMEM, L15, SkBM, and
RITC80-7. Many of these basal culture media are commercially
available, and their compositions are known.
The basal culture medium may be used as is in a standard
composition (for example, in an as-commercialized state) or may be
used after its composition is appropriately modified according to
the cell species or cell conditions. Therefore, the basal culture
medium for use in the present disclosure is not limited to those of
known compositions but include those obtained by addition or
removal of one or more components or by increasing or decreasing
the amounts of one or more components.
The cultivation of the cell population can be carried out under
conditions ordinarily adopted in the technical field concerned. For
example, a typical cultivation condition resides in conducting
cultivation at 37.degree. C. and 5% CO2. The cultivation can be
performed in a vessel of any size and shape. The size and shape of
the sheet-shaped cell culture can be arbitrarily controlled by
controlling the size and shape of the cell adhesion surface of the
culture vessel, or by disposing a frame of desired size and shape
on the cell adhesion surface of the culture vessel and culturing
the cells inside the frame.
The sheet-shaped cell culture production method of the present
disclosure may further include a step of recovering the
sheet-shaped cell culture. The recovery of the sheet-shaped cell
culture is not particularly limited, so long as the sheet-shaped
cell culture can be freed (exfoliated) from the culture substrate
serving as a scaffold while maintaining its sheet structure at
least partly. For example, the recovery can be carried out by an
enzyme treatment using a proteinase (for example, trypsin) and/or a
mechanical treatment such as pipetting. In addition, in the case
where the sheet-shaped cell culture is formed by culturing a cell
population on a culture substrate having a surface coated with a
material whose property is varied in response to a stimulus, such
as temperature or light, the sheet-shaped cell culture can be
non-enzymatically freed by giving a predetermined stimulus.
A preferred embodiment of the sheet-shaped cell culture production
method of the present disclosure includes the following steps:
(1) a step of changing a character of contaminating cells in a cell
population including target cells and the contaminating cells that
is collected from a subject;
(2) a step of obtaining a cell population with a high target cell
purity by removing the contaminating cells and/or collecting the
target cells based on the changed character;
(3) a step of freezing the cell population;
(4) a step of thawing the frozen cell population;
(5) a step of culturing the cell population into a sheet shape to
form a sheet-shaped cell culture; and
(6) a step of recovering the sheet-shaped cell culture formed.
The steps of (1), (2), (5) and (6) are the same as described above.
In the above embodiment, the target cells are preferably myoblasts
(particularly, skeletal myoblasts) or mesenchymal stem cells, and
the contaminating cells are preferably fibroblasts.
The step (3) can be carried out by any known technique. Examples of
such a technique include, without restriction, serving the cell
population in a vessel to freezing means, for example, a freezer, a
deep freezer, or a low-temperature medium (for example, liquid
nitrogen). The temperature of the freezing means is not
particularly limited, so long as it is a temperature at which part
of the cell population in the vessel, preferably the whole of the
cell population, can be frozen. Typically, the temperature is, for
example, 0.degree. C. or below, preferably -20.degree. C. or below,
more preferably -40.degree. C. or below, and further preferably
-80.degree. C. or below. In addition, the cooling rate in the
freezing operation is not particularly limited, so long as the
cooling rate does not largely impair the survival rate or functions
of the cells upon freezing and thawing. Typically, the cooling rate
is such that cooling from, for example, 4.degree. C. to -80.degree.
C. takes one to five hours, preferably two to four hours, and more
particularly approximately three hours. Specifically, the cooling
can be performed at a rate of, for example, 0.46.degree. C./minute.
Such a cooling rate can be achieved by a method wherein the vessel
containing the cell population is served, either directly or in the
state of being contained in a freezing treatment vessel, to the
freezing means set at a desired temperature. The freezing treatment
vessel may have a function to control the lowering rate of the
temperature inside the vessel to a predetermined rate. As such a
freezing treatment vessel, there can be used any known one, for
example, BICELL.RTM. (Nihon Freezer Co., Ltd.).
The freezing operation may be conducted with the cell population
kept immersed in a culture liquid or a physiological buffer
solution. In addition, the freezing operation may be performed
after a treatment such as addition of a cryoprotective agent for
protecting the cells from freezing and thawing operations to the
culture liquid, or replacement of the culture liquid with a
cryopreservation liquid containing a cryoprotective agent.
Therefore, the sheet-shaped culture production method of the
present disclosure may further include a step of adding a
cryoprotective agent to a culture liquid, or a step of replacing
the culture liquid with a cryopreservation liquid. In the case of
replacing the culture liquid with the cryopreservation liquid, the
cryopreservation liquid may be added after removing the culture
liquid substantially completely or may be added while part of the
culture liquid is kept remaining, if the cryoprotective agent is
contained in an effective concentration in the liquid in which the
cells are immersed at the time of freezing. Here, the "effective
concentration" means a concentration at which the cryoprotective
agent, while showing no toxicity, shows a cryoprotective effect,
for example, an effect of inhibiting a lowering in the survival
rate, vitality, functions or the like of the cells upon freezing
and thawing, as compared to the case of not using the
cryoprotective agent. Such a concentration is known to persons
skilled in the art, or can be appropriately determined through
routine experiments.
The cryoprotective agent is not specifically restricted, so long as
it shows a cryoprotective effect on cells. Examples of the
cryoprotective agent include dimethyl sulfoxide (DMSO), glycerol,
ethylene glycol, propylene glycol, sericin, propanediol, dextran,
polyvinyl pyrrolidone, polyvinyl alcohol, hydroxyethyl starch,
chondroitin sulfate, polyethylene glycol, formamide, acetamide,
adonitol, perseitol, raffinose, lactose, trehalose, sucrose, and
mannitol. The cryoprotective agents may be used either singly or in
combination of two or more of them.
The addition concentration of the cryoprotective agent added to the
culture liquid or the concentration of the cryoprotective agent in
the cryopreservation liquid is not particularly limited, so long as
the concentration is the effective concentration as defined above.
Typically, the concentration is, for example, 2 to 20% (v/v) to the
whole part of the culture liquid or the cryopreservation liquid.
However, those alternative use concentrations which fall out of
this concentration range but which are known for the respective
cryoprotective agents or are determined empirically can also be
adopted. Such alternative concentrations are also within the scope
of the present disclosure.
The step (4) can be performed by any known cell thawing means.
Typically, the step (4) can be achieved, for example, by a method
wherein the frozen cell population is served to thawing means, for
example, a solid, liquid or gaseous medium (for example, water), a
water bath, an incubator, or a thermostat at a temperature higher
than the freezing temperature, or by a method wherein the frozen
cell population is immersed in a medium (for example, culture
medium) at a temperature higher than the freezing temperature,
these methods being non-limitative. The temperature of the thawing
means or the immersion medium is not particularly limited, so long
as it is a temperature at which the cell population can be thawed
within a desired period of time. Typically, for example, the
temperature is 4.degree. C. to 50.degree. C., preferably 30.degree.
C. to 40.degree. C., and more preferably 36.degree. C. to
38.degree. C. In addition, the thawing time is not specifically
restricted so long as it does not largely impair the survival rate
or functions of the cells upon thawing. The thawing time is
typically within, for example, two minutes; particularly, when the
thawing time is within 20 seconds, lowering in the survival rate or
functions can be largely restrained. The thawing time can be
controlled, for example, by changing the temperature of the thawing
means or immersion medium, the volume or composition of the culture
liquid or cryopreservation liquid at the time of freezing.
The sheet-shaped cell culture production method as above may
include a step of washing the cell population, between the step (4)
and the step (5). The washing of the cell population can be carried
out by any known technique. Typically, the washing is achieved, for
example, by suspending the cell population in a liquid (for
example, a culture liquid, or a physiological buffer solution that
contains, or does not contain, serum or serum component (serum
albumin or the like)), subjecting the suspension to centrifugation,
discarding the supernatant liquid, and recovering the precipitated
cell population, this method being non-limitative. In the step of
washing the cell population, such a cycle of suspension,
centrifugation and recovery may be performed once or multiple times
(for example, two, three, four, or five times). In one embodiment
of the present disclosure, the step of washing the cell population
is carried out immediately after the step (4) of thawing the frozen
cell population.
In one embodiment, all the steps of the production method
(inclusive of the cell population production method and the
sheet-shaped cell culture production method) of the present
disclosure are carried out in vitro. In another embodiment, the
production method of the present disclosure includes a step which
is carried out in vivo and examples of which include, without
restriction, the step of collecting a cell population or a tissue
as a source of a cell population from a subject. In one embodiment,
all the steps of the production method of the present disclosure
are carried out under aseptic conditions. In one embodiment, the
production method of the present disclosure is performed in such a
manner that the cell population or sheet-shaped cell culture
obtained finally will be substantially aseptic. In one embodiment,
the production method of the present disclosure is carried out in
such a manner that the cell population or sheet-shaped cell culture
obtained finally will be aseptic.
Another aspect of the present disclosure relates to a sheet-shaped
cell culture containing the high-purity cell population of the
present disclosure (hereinafter, such a sheet-shaped cell culture
may be referred to simply as "the sheet-shaped cell culture").
The sheet-shaped cell culture of the present disclosure is high in
target cell purity. The extent of highness in purity is as
described above in regard of the high-purity cell population of the
present disclosure. The target cells are preferably myoblasts,
particularly skeletal myoblasts, or mesenchymal stem cells. The
sheet-shaped cell culture of the present disclosure may be one that
is produced by the sheet-shaped cell culture production method of
the present disclosure. The sheet-shaped cell culture of the
present disclosure is preferably aseptic. The sheet-shaped cell
culture of the present disclosure contains the target cells at high
purity, and, therefore, is higher in therapeutic effect or the
like, as compared to sheet-shaped cell cultures, which do not
contain the high-purity cell population of the present
disclosure.
The high-purity cell population and sheet-shaped cell culture of
the present disclosure are useful for treatment of various
diseases, particularly diseases associated with tissue abnormality.
In one embodiment, therefore, the high-purity cell population and
sheet-shaped cell culture of the present disclosure are for use in
treating diseases associated with tissue abnormality. Examples of
the tissue to be treated include, without restriction, a cardiac
muscle, a cornea, a retina, an esophagus, skin, a joint, cartilage,
a liver, a pancreas, gingiva, a kidney, a thyroid gland, a skeletal
muscle, a middle ear, and bone marrow. In addition, the diseases to
be treated can include, without restriction, cardiac diseases (for
example, myocardial damage (myocardial infarction, cardiac injury),
cardiomyopathy, etc.), corneal diseases (for example, corneal
epithelial stem cell deficiency, corneal injury (thermal/chemical
corrosion), corneal ulcer, corneal clouding, corneal trepanation,
corneal cicatrization, Stevens-Johnson syndrome, ocular pemphigoid,
etc.), retinal diseases (for example, retinitis pigmentosa, and
age-related macular degeneration, etc.), esophageal diseases (for
example, prevention of inflammation or stenosis of esophagus after
esophageal surgery (removal of esophageal cancer), etc.), skin
diseases (for example, skin injury (traumatic injury and burn),
etc.), joint diseases (for example, degenerative arthritis, etc.),
cartilage diseases (for example, cartilage injury, etc.), liver
diseases (for example, chronic hepatopathy, etc.), pancreatic
diseases (for example, diabetes, etc.), dental diseases (for
example, periodontal disease, etc.), renal diseases (for example,
renal insufficiency, renal anemia, renal osteodystrophy, etc.),
thyroid diseases (for example, hypothyrosis, etc.), muscular
diseases (for example, muscle injury, myositis, etc.), middle ear
diseases (for example, tympanitis, etc.), and bone marrow diseases
(for example, leukemia, aplastic anemia, immunodeficiency disease,
etc.).
That the high-purity cell population and sheet-shaped cell culture
of the present disclosure are useful for the above-mentioned
diseases is described in, for example, Patent Document 1,
Non-patent Document 1, Arauchi et al., Tissue Eng Part A. 2009
December; 15(12): 3943-9, Ito et al., Tissue Eng. 2005 March-April;
11(3-4): 489-96, Yaji et al., Biomaterials. 2009 February; 30(5):
797-803, Yaguchi et al., Acta Otolaryngol. 2007 October; 127(10):
1038-44, Watanabe et al., Transplantation. 2011 Apr. 15; 91(7):
700-6, Shimizu et al., Biomaterials. 2009 October; 30(30): 5943-9,
Ebihara et al., Biomaterials. 2012 May; 33(15): 3846-51, Takagi et
al., World J Gastroenterol. 2012 Oct. 7; 18(37): 5145-50, etc.
While the high-purity cell population and sheet-shaped cell culture
of the present disclosure can be applied to a tissue to be treated
and can thereby be used for repairing or regenerating the tissue,
they can also be transplanted to other part (for example,
subcutaneous tissue) than the tissue to be treated, as a source of
a biologically active agent such as hormone (for example, Arauchi
et al., Tissue Eng Part A. 2009 December; 15(12): 3943-9, Shimizu
et al., Biomaterials. 2009 October; 30(30): 5943-9, etc.). In
addition, the sheet-shaped cell culture of the present disclosure
can be fragmented to an injectable size and the sheet-shaped cell
culture can be injected into a part needing a treatment (Wang et
al., Cardiovasc Res. 2008 Feb. 1; 77(3): 515-24).
The high-purity cell population and sheet-shaped cell culture of
the present disclosure may further contain various additive
components, examples of which include a carrier that is
pharmaceutically acceptable, components for enhancing the
viability, engrafting property and/or functions of the high-purity
cell population and/or sheet-shaped cell culture, and other
effective components useful for treatment of the disease to be
treated. As such additive components, there can be used any known
ones, and persons skilled in the art are well informed about these
additive components. In addition, the high-purity cell population
and sheet-shaped cell culture of the present disclosure can be used
in combination with a component for enhancing the viability,
engrafting property and/or functions of the high-purity cell
population and/or sheet-shaped cell culture, or other effective
component useful for treatment of the disease to be treated.
A further aspect of the present disclosure relates to a medical
composition containing an effective component selected from the
group including the high-purity cell population of the present
disclosure and the sheet-shaped cell culture of the present
disclosure.
The medical composition of the present disclosure may contain, in
addition to the high-purity cell population and/or sheet-shaped
cell culture of the present disclosure, various additive
components, examples of which include a carrier that is
pharmaceutically acceptable, components for enhancing the
viability, engrafting property and/or functions of the high-purity
cell population and/or sheet-shaped cell culture, and other
effective components useful for treatment of the disease to be
treated. As such additive components, there can be used any known
ones, and persons skilled in the art are well acquainted with these
additive components. IN addition, the medical composition of the
present disclosure can be used jointly with a component for
enhancing the viability, engrafting property and/or functions of
the high-purity cell population and/or sheet-shaped cell culture,
or other effective component useful for treatment of the disease to
be treated. In one embodiment, the medical composition of the
present disclosure is for use in treating diseases associated with
tissue abnormality. The tissues and diseases to be treated are as
described above in relation to the high-purity cell population and
sheet-shaped cell culture of the present disclosure.
Another aspect of the present disclosure relates to a kit (set or
pack) which includes part or all of the elements to be used for
production of the high-purity cell population, sheet-shaped cell
culture or composition (for example, medical composition) of the
present disclosure and which is for producing the high-purity cell
population, sheet-shaped cell culture or composition (for example,
medical composition) of the present disclosure, or for treating a
disease (for example, diseases associated with tissue abnormality)
(hereinafter, such a kit may be referred to as "the production kit
of the present disclosure"). Herein, the terms "set" and "pack" are
used interchangeably with "kit" and, hence, the descriptions
related to the "kit" herein also apply to the "set" and the
"pack."
Specific elements included in the kit of the present disclosure
will be made clear by reference to the above descriptions related
to the production of the high-purity cell population, sheet-shaped
cell culture or medical composition or the like of the present
disclosure, and, therefore, not all the elements are set forth
here. In one embodiment, the kit of the present disclosure may
include various elements, examples of which include, without
restriction, an agent for changing a character of the contaminating
cells (for example, an agent for conversion of cell species, an
agent for marker expression or for inhibition of marker expression,
an agent for conversion to iPS cells, an agent for inducing
differentiation of iPS cells to other cell species, etc.), a cell
population for use in production of the high-purity cell population
(for example, a cell population isolated from a living body, etc.),
cells for use in cultivating the sheet-shaped cell culture (for
example, the high-purity cell population of the present disclosure,
etc.), a culture liquid, a culture dish, a washing liquid, an agent
for use in purification of the target cells and/or removal of the
contaminating cells (for example, an antibody, a washing liquid,
etc.), instruments (for example, pipette, dropping pipette,
tweezers, beads, affinity column, filter, etc.), and instructions
related to the production method and/or use method of the
high-purity cell population, sheet-shaped cell culture or
composition or the like (for example, an instruction book, a medium
such as flexible disc, CD, DVD, Blu-ray Disc, memory card or USB
memory on which information about the production method and/or use
method is recorded, etc.).
In a specific embodiment, the kit for producing the high-purity
cell population or composition containing the same of the present
disclosure includes an agent for changing a character of the
contaminating cells, and an agent for use in purification of the
target cells and/or removal of the contaminating cells. In another
specific embodiment, the kit for producing the high-purity cell
population or composition containing the same of the present
disclosure includes a cell population isolated from a living body,
an agent for changing a character of the contaminating cells, and
an agent for use in purification of the target cells and/or removal
of the contaminating cells. In a further embodiment, the kit for
producing the sheet-shaped cell culture or composition containing
the same of the present disclosure includes the high-purity cell
population of the present disclosure, a culture liquid and a
culture dish. In another embodiment, the kit for producing the
sheet-shaped cell culture or composition containing the same of the
present disclosure includes a cell population isolated from a
living body, an agent for changing a character of the contaminating
cells, an agent for use in purification of the target cells and/or
removal of the contaminating cells, a culture liquid and a culture
dish.
A further aspect of the present disclosure relates to a method for
treating a subject's disease, the method including a step of
administering an effective amount of the high-purity cell
population, sheet-shaped cell culture or medical composition of the
present disclosure to the subject needing the same (hereinafter,
such a method may be referred to simply as "treatment method"). The
tissue and disease to be treated by the treatment method of the
present disclosure are as described above in relation to the
high-purity cell population and sheet-shaped cell culture of the
present disclosure. In addition, in the treatment method of the
present disclosure, components for enhancing the viability,
engrafting property and/or functions or the like of the high-purity
cell population and/or sheet-shaped cell culture, other effective
components useful for treatment of the disease to be treated, or
the like can be used jointly with the high-purity cell population,
sheet-shaped cell culture or medical composition of the present
disclosure.
The treatment method of the present disclosure may further include
a step of producing the high-purity cell population, sheet-shaped
cell culture or medical composition according to the production
method of the present disclosure. The treatment method of the
present disclosure may further include a step of collecting the
whole blood, cells or a tissue as a source of cells for production
of the high-purity cell population from a subject, prior to the
step of producing the high-purity cell population, sheet-shaped
cell culture or medical composition. In one embodiment, the subject
from whom the whole blood and/or cells or a tissue as a source of
cells is collected is the same individual as the subject to whom
the high-purity cell population, sheet-shaped cell culture or
medical composition is to be administered. In another embodiment,
the subject from whom the whole blood and/or cells or a tissue as a
source of cells is collected is the same species as, but is a
different individual from, the subject to whom the high-purity cell
population, sheet-shaped cell culture or medical composition is to
be administered. In a further embodiment, the subject from whom the
whole blood and/or cells or a tissue as a source of cells is
collected is an individual of a different species from the subject
to whom the high-purity cell population, sheet-shaped cell culture
or medical composition is to be administered.
In the present disclosure, the subject may be healthy or may be
suffering from some disease. In the case where it is planned to
treat a disease, the subject typically means a subject suffering
from the disease or a subject having a risk of contracting the
disease.
In addition, the term "treatment" includes all kinds of medically
acceptable preventive and/or curative interventions aimed at cure,
temporary remission or prevention of a disease. For instance, the
term "treatment" includes medically acceptable interventions for
various purposes, such as slowing down or stopping the progress of
a disease, causing retraction or disappearance of a lesion, and
preventing onset of a disease or preventing recurrence of a
disease.
In the present disclosure, an effective amount refers, for example,
to an amount (for example, the number of cells contained in the
high-purity cell population, sheet-shaped cell culture or medical
composition, the size or weight of the sheet-shaped cell culture)
making it possible to inhibit onset or recurrence of a disease, to
alleviate a symptom, or to slow down or stop the disease
progression, and is preferably such an amount as to prevent the
onset or recurrence of the disease or to cure the disease. In
addition, the effective amount is preferably such an amount as not
to produce an adverse influence in excess of the advantageous
effect of administration. Such an amount can be appropriately
determined by, for example, tests performed using laboratory
animals or disease model animals such as mice, rats, dogs and pigs,
and the methods for such tests are well known to those skilled in
the art. Besides, the size of a tissue lesion to be treated can be
an important index for determination of the effective amount.
The high-purity cell population, sheet-shaped cell culture or
medical composition of the present disclosure can be administered
by way of a variety of routes such as intravenous, intramuscular,
subcutaneous, local, intraarterial, intraportal, intraventricular,
and intraperitoneal routes. In the case of the sheet-shaped cell
culture or medical composition containing the same, the
administration method may be, for example, direct application to
the tissue. In addition, in the case of using fragments of the
sheet-shaped cell culture, the fragments may be administered by way
of various routes suited to administration by injection, such as
intravenous, intramuscular, subcutaneous, local, intraarterial,
intraportal, intraventicular, and intraperitoneal routes.
The frequency of administration is typically once per treatment;
when this is not enough to obtain a desired effect, however, the
administration may be carried out multiple times.
Example 1
Purification of Skeletal Myoblasts by Conversion of Fibroblasts
into Fat Cells
A cell suspension of a cell population isolated from a skeletal
muscle is seeded onto a cell culture plate in a quantity of
0.8.times.105 cells/well, and is cultured overnight in a CO2
incubator. In a microtube is placed 30 .mu.L of a serum-free
culture medium, and 1.0 .mu.g of PPAR.gamma. expression adenovirus
vector (AdRGD-PPAR.gamma., Gastroenterology. 2003 May; 124(5):
1315-24) is added thereto. The contents of the microtube are mixed
by pipetting, then 5 .mu.L of HilyMax solution (manufactured by
Dojindo Laboratories) is added thereto, and the system is let stand
still at room temperature for 15 minutes, to prepare an
AdRGD-PPAR.gamma.-HilyMax complex. The AdRGD-PPAR.gamma.-HilyMax
complex is added to the cultured cell population, and the plate is
shaken gently. The cells are cultured in a CO2 incubator for 24
hours. The cell population is washed with PBS, followed by
centrifugation, after which the cell population is seeded into a
80-cm2 flask in a density of 2 to 3.times.104 cells/cm2, and is
cultured by use of 15 mL of culture medium (fat cell
differentiation-inducing medium, manufactured by Cosmo Bio Co.,
Ltd.) for five to seven days. After the cultivation, the culture
surface is washed with PBS, followed by a trypsin treatment, to
obtain a cell population, which is filtered through a mesh (cell
strainer, manufactured by Corning Incorporated) having a pore
diameter of 70 .mu.m, whereby the cell population having passed
through the mesh is obtained as the desired cell population. The
cell population thus obtained is labelled with anti-CD56 antibody,
and the proportion of CD56-positive cells (skeletal myoblast
purity) is measured by use of a flow cytometer.
The detailed description above describes a method of producing a
cell population with a high target cell purity, a cell population
obtained by the method, and a medical composition containing the
cell population. The invention is not limited, however, to the
precise embodiments and variations described. Various changes,
modifications and equivalents can effected by one skilled in the
art without departing from the spirit and scope of the invention as
defined in the accompanying claims. It is expressly intended that
all such changes, modifications and equivalents which fall within
the scope of the claims are embraced by the claims.
* * * * *